Methods for preparing 2-alkynyladenosine derivatives

ABSTRACT

Disclosed are methods for preparing 2-alkynyladenosine derivatives of formula A:  
                 
or a stereoisomer, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate or isomorphic crystalline form thereof, the method comprising the step of: 
 
contacting 2-iodoadenosine-5′-N-ethyluronamide with a compound of formula B:  
                 
         wherein Z is —C(═O)OR or —CH 2 OC(═O)R, where R is a C 1  to C 5  alkyl, preferably methyl. The methods are useful for preparing 2-alkynyladenosine derivatives that are, in certain embodiments, adenosine receptor agonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Application Ser. No. 60/471,643,filed May 19, 2003, the disclosure of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to improved methods for preparing2-alkynyladenosine derivatives, more specifically, to improved methodsfor preparing 2-alkynyladenosine derivatives that are, in certainembodiments, adenosine receptor agonists and, even more specifically, toimproved methods for preparing 2-alkynyladenosine derivatives that are,in certain embodiments, A₂ adenosine receptor agonists.

BACKGROUND OF THE INVENTION

Adenosine is known to modulate a number of physiological functions. Atthe cardiovascular system level, adenosine is a strong vasodilator and acardiac depressor. In the central nervous system, adenosine inducessedative, anxiolytic and antiepileptic effects. At the kidney level, itexerts a diphasic action, inducing vasoconstriction at lowconcentrations and vasodilation at high doses. Adenosine acts as alipolysis inhibitor on fat cells and as an anti-aggregant on platelets(Stone T. W., Purine Receptors and their Pharmacological Roles, Advancesin Drug Research, Academic Press Limited, 1989, 18, 291-429; ProgressCardiovasc. Dis. 1989, 32, 73-97).

A number of studies have shown that adenosine actions are mediated bytwo subtypes of receptors that are located on the cell membrane: one ofhigh-affinity, inhibiting the activity of the enzyme adenylate cyclase(A₁ receptor), and another of low-affinity, stimulating the activity ofthe same enzyme (A₂ receptor). (J. Med. Chem. 1982, 25, 197-207;Physiol. Rev. 1990, 70(3), 761-845; J. Med. Chem. 1992, 35, 407-422).Both receptors are widely spread in the different systems of theorganism. In some tissues, however, only one of said receptors is mainlypresent. For example, the A₁ receptor is more prevalent than the A₂receptor at the cardiac level, whereas the A₂ receptor is more prevalentthan the A₁ receptor at the vascular level and on platelets.

Compounds capable of interacting selectively with either the A₁ or A₂receptor could have an interesting pharmacological pattern. Furthermore,the vasodilating activity, together with the anti-aggregating action, ofthe compounds that interact with the A₂ receptors may lead to usefultherapeutic applications in the treatment of severe cardiovascularpathologies, such as ischemic cardiopathy, hypertension andatherosclerosis. Moreover, due to the actions on central nervous system,the use of A₂ selective medicaments can be envisaged in the treatment ofcerebrovascular ischemia, epilepsy and various emotional disorders, suchas anxiety and psychosis.

Previously, adenosine-5′-N-ethyluronamide or NECA (Mol. Pharmacol.,1986, 25, 331-336) was the only known compound, other than adenosine,having agonist activity at the A₂ receptor. Unfortunately, NECA is alsoactive on the A₁ receptor and thus lacks specificity for the A₂receptors alone. Because it was the only available compound having A₂affinity, NECA was used for pharmacological tests for the receptorbinding.

More recently, however, certain NECA derivatives having A₂ receptorselectivity have been developed. These compounds are NECA derivativesthat are substituted at the C2-position with phenylamino groups. Forexample, the compound2-(p-(carboxyethyl)phenylethylamino)-5′-N-ethyluronamide, named CGS21680 (J. Pharmacol Exp. Ther., 1989, 251, 888-893), has become thereference compound for the pharmacological studies on A₂ receptor.

Other purine derivatives having selective A₂ agonist activity aredisclosed, for example, in GB-A-2203149; EP-A-0309112; EP-A-0267878;EP-A-0277917; and EP-A-0323807. Substitution at the 2-position of thepurine group has been considered promising to give the desiredselectivity (J. Med. Chem. 1992, 35, 407-422). 2-Alkynylpurinederivatives have been disclosed in EP-A-0219876 and U.S. Pat. No.4,956,345.

U.S. Pat. No. 5,593,975 discloses 2-alkynyl adenosine derivativessubstituted at the ethyne position with aryl, heterocyclic orhydroxyalkyl groups and in which the riboside residue is substituted bythe N-alkyl- (or cycloalkyl)-uronamido. It is reported that thesecompounds exhibit strong A₂ agonist selectivity and, therefore, areuseful for the treatment of cardiovascular pathologies, such as cardiacischemia, hypertension and atherosclerosis and of diseases of thecentral nervous system, such as cerebrovascular ischemia, epilepsy andemotional disorders (anxiety and psychosis).

The 2-alkynyl adenosine derivatives of U.S. Pat. No. 5,593,975 have thefollowing general formula:

-   -   wherein R is hydrogen, C₁-C₆ alkyl, C₃-C₇ cycloalkyl, phenyl        C₁-C₃ alkyl;    -   wherein R₁ has one of the following meanings:    -   (a) phenyl or naphthyl optionally substituted with one to three        halogen atoms (chlorine, fluorine or bromine), C₁-C₆ alkyl,        C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆        alkoxycarbonyl, C₂-C₆ alkoxyalkyl, C₁-C₆ alkylthio, thio, CHO,        cyanomethyl, nitro, cyano, hydroxy, carboxy, C₂-C₆ acyl, amino,        C₁-C₃ monoalkylamino, C₂-C₆ dialkylamino, methylenedioxy; or        aminocarbonyl;    -   (b) a group of the formula —(CH₂)_(m)-Het wherein m is 0 or an        integer from 1 to 3 and Het is 5 or 6 membered heterocyclic        aromatic or non aromatic ring, optionally benzocondensed,        containing 1 to 3 heteroatoms selected from oxygen, nitrogen or        sulphur, linked through a carbon atom or through a nitrogen        atom;    -   (c) C₃-C₇ cycloalkyl optionally containing unsaturations or        C₂-C₄ alkenyl;    -   (d) moieties of the following formula:    -    where R₂ is hydrogen, methyl or phenyl;        -   -   R₄ is OH, NH₂, dialkylamino, halogen, or cyano;            -   R₅ is hydrogen, C₁-C₆ linear or branched alkyl, C₅-C₆                cycloalkyl or C₃-C₇ cycloalkenyl, phenyl- C₁-C₂-alkyl            -   or R₂ and R₅, taken together, form a 5 or 6-membered                carbocyclic ring or R₃ is hydrogen and R₂ and R₄, taken                together, form an oxo group or a corresponding acetalic                derivative;            -   when R is different from hydrogen and/or R₃ is different                from ethyl, R₁ can also be C₁-C₆ linear or branched                alkyl; and            -   n is 0 or 1 to 4; and

        -   wherein R₃ is C₁-C₆ alkyl, C₃-C₇-cycloalkyl, phenyl or            benzyl; provided that when R is different that hydrogen or            when R is hydrogen and R₃ is cyclopentyl, phenyl or benzyl,            R₁ can also be C₁-C₆ linear or branched alkyl.

The 2-alkynyl adenosine compounds of U.S. Pat. No. 5,593,975 areprepared by the general synthetic schemes shown below.

In schemes I and II, R′ and R′₁ have the same meanings as R and R₁,respectively, or they are groups which can be converted into R and R₁,respectively, for example, by removing any protecting groups which canbe present in R′ and R′₁ compatible with the reaction conditions; Y isBr or I and X is chlorine, bromine or iodine.

The reactions shown in schemes I and II are carried out in the presenceof catalysts (for example: bis(triphenylphosphine) palladium dichlorideand a cuprous halide) and a suitable acid-binding agent, such as anorganic base (for example: triethylamine, diisopropylethylamine orpyridine).

As the solvent, a substituted amide (such as dimethylformamide), anether (such as dioxane or tetrahydrofuran), acetonitrile or optionally amixture of two or more of said solvents, are preferably used.

The compounds of formula II, in which Y is iodine and R′ is hydrogen,can be prepared from 2-iodoadenosine (Nair et al., Synthesis, 1982,670-672) according to the following Scheme III:

Compounds of formula VIII in which Y is iodine and R′ is different fromhydrogen can be prepared according to the following Scheme IV:

In the above Scheme IV, R, R₁ and R₃ are as above defined.

The compounds of formula V are prepared by reaction of compounds offormula II with an acetylene derivative, for example,1-trimethylsilylacetylene, under the conditions reported for thereaction between compounds II and III. Compounds III and IV are known orthey can be prepared according to well-known methods.

U.S. Pat. No. 6,322,771 discloses compounds of formula IX:

wherein

-   -   (a) each R is individually hydrogen, C₁-C₆ alkyl, C₃-C₇        cycloalkyl, phenyl or phenyl(C₁-C₃)-alkyl;    -   (b) X is —CH₂OH, —CO₂R², —OC(O)R², —CH₂OC(O)R² or —C(O)NR³R⁴;    -   (c) each of R², R³ and R⁴ is individually H, C₁-C₆-alkyl;        C₁-C₆-alkyl substituted with 1-3 C₁-C₆ alkoxy, C₃-₇cycloalkyl,        C₁-C₆-alkylthio, halogen, hydroxy, amino,        mono(C₁-C₆-alkyl)amino, di(C₁-C₆-alkyl)amino, or C₆-C₁₀-aryl,        wherein aryl may be substituted with 1-3 halogen, C₁-C₆-alkyl,        hydroxy, amino, mono(C₁-C₆-alkyl)amino, or di(C₁-C₆-alkyl)amino;        C₆-C₁₀-aryl; or C₆-C₁₀-aryl substituted with 1-3 halogen,        hydroxy, amino, mono(C₁-C₆-alkyl)amino, di(C₁-C₆-alkyl)amino, or        C₁-C₆-alkyl; and    -   (d) R¹ is (X—(Z)—)_(n)[(C₃-C₁₀)cycloalkyl]—(Z′)—wherein Z and Z′        are individually (C₁-C₁₀)alkyl, optionally interrupted by 1-3 S        or nonperoxide O, or is absent, and n is 1-3.        It is disclosed that the compounds of Formula IX may be prepared        by the synthetic methods disclosed in U.S. Pat. Nos. 5,278,150;        5,140,015; 5,877,180; 5,593,975; and 4,956,345.

U.S. Pat. No. 6,322,771 discloses preferred compounds of Formula IX,wherein each R is H, X is ethylaminocarbonyl and R¹ is4-carboxycyclohexylmethyl (DWH-146a), R¹ is4-methoxycarbonylcyclohexylmethyl (DWH-146e) or R¹ is4-acetoxymethyl-cyclohexylmethyl (JMR-193):

According to U.S. Pat. No. 6,322,771, the synthesis of the methyl4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3,4-dihydroxytetrahydro-Z-furanyl-9H-2-purinyl)-2-propynyl]-1-cyclohexanecarboxylate(DWH-146e) was accomplished by the cross coupling of an iodo-adenosinederivative(N-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide)with methyl 4-(2-propynyl)-1-cyclohexanecarboxylate by utilization of aPd(II) catalyst.

The iodo-adenosine derivative was first prepared from guanosine bytreating it with acetic anhydride, which acetylates the sugar hydroxyls.The resulting compound was then chlorinated at position 6 withtetramethyl amimonium chloride and phosphorous oxychloride. Iodinationof position 2 was accomplished via a modified Sandmeyer reaction,followed by displacement of the 6-C1 and sugar acetates with ammonia.The 2′ and 3′ hydroxyls were protected as the acetonide and the 5′hydroxyl was oxidized to the acid with potassium permanganate.Deprotection of the 2′ and 3′ acetonide, Fisher esterification of the 5′acid with ethanol and conversion of the resulting ethyl ester to theethyl amide with ethylamine gaveN-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramide.

The acetylene [methyl 4-(2-propynyl)-1-cyclohexanecarboxylate] wassynthesized starting from trans-1,4-cyclohexanedimethanol. Initially,the trans-diol was monotosylated followed by displacement of thetosylate with an acetylene anion. The hydroxyl of the resulting hydroxylacetylene species was oxidized to the acid via Jones reagent followed bymethylation with (trimethylsilyl)diazomethane to give methyl4-(2-propynyl)-1-cyclohexanecarboxylate.

A cross-coupling reaction ofN-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D-ribofuranuoramideand methyl 4-(2-propynyl)-1-cyclohexanecarboxylate was then performed.To a solution of N,N-dimethylformamide (0.5 mL), acetonitrile (1 mL),triethylamine (0.25 mL), andN-ethyl-1′-deoxy-1′-(amino-2-iodo-9H-purin-9-yl)-β-D -ribofuranuroamide(25 mg, 0.06 mmol) was added bis(triphenylphosphine)palladium dichloride(1 mg, 2 mol %) and copper(I)iodide (0.06 mg, 0.5 mol %). To theresulting mixture was added methyl4-(2-propynyl)-1-cyclohexanecarboxylate (54 mg, 0.3 mmol) and thereaction was stirred under nitrogen atmosphere for 16 hours. The solventwas removed under vacuum and the resulting residue was flashchromatographed in 20% methanol in chloroform (R_(f)=0.45) to give 19 mg(off-white solid, mp 125° C. (decomposed)) of4[3-(6-amino-9(5-[(ethylamino)carbonyl]-3,4-dihydroxytetrahydro-Z-furanyl)-9H-2-purinyl)-2-propynyl]-1-cyclohexanecarboxylate(DWH-146e).

These above-described synthetic methods for producing 2-alkynyladenosinederivatives, including DWH-146e, provide lower yields than desired,require prolonged reaction times and require extensive chromatographicpurification. For example in U.S. Pat. No. 5,593,975, the acetonideprotection (first step of Scheme III above) requires purification bycolumn chromatography. Furthermore, the oxidation procedure (second stepof Scheme III above) requires prolonged reaction times and has beennoted to be troublesome due to competing oxidation at the 2-iodoposition. Homma et al., J. Med. Chem., 1992, 35, 2881-2890. In U.S. Pat.No. 6,322,771, the first step of the synthesis of thecyclohexane-containing acetylene requires purification by columnchromatography to separate the desired mono-tosyl product from thestarting diol and bis-tosyl products. In the second step of thesynthesis of the cyclohexane-containing acetylene, prolonged reactiontimes, a large excess of the acetylene anion and purification by columnchromatography are required. Furthermore, the cross-coupling reactionbetween the cyclohexane-containing acetylene and 2-iodoNECA proceedswith poor yield after chromatography.

Thus, there is a need for improved synthetic methods for producing2-alkynyladenosine derivatives that provide higher yields and requireless purification than prior art methods. Using alternative reagents andsteps, we have discovered an improved method for producing2-alkynyladenosine derivatives at higher yields with lesschromatographic purification required.

SUMMARY OF THE INVENTION

In one embodiment, the invention is directed to methods for preparingcompounds of formula A:

or a stereoisomer, pharmaceutically acceptable salt, hydrate, solvate,acid salt hydrate or isomorphic crystalline form thereof the methodcomprising the step of:

-   -   contacting 2-iodoadenosine-5′-N-ethyluronamide with a compound        of formula B:    -   wherein Z is —C(═O)OR or —CH₂OC(═O)R, where R is a C₁ to C₅        alkyl, preferably methyl.

The invention is directed, inter alia, to improved synthetic methods forproducing 2-alkynyladenosine derivatives that provide higher yields andrequire less chromatographic purification than prior art methods, usingalternative reagents and steps.

In another embodiment, the invention is directed to methods forpreparing 2-iodoadenosine-5′-N-ethyluronamide, comprising the steps of:

-   -   providing 2-iodoadenosine;    -   protecting the hydroxyl groups of said 2-iodoadenosine with an        acetonide group;    -   oxidizing the primary alcohol of said acetonide-protected        2-iodoadenosine to an acid derivative of said        acetonide-protected 2-iodoadenosine;    -   converting said acid derivative to an N-ethylamide derivative of        said acetonide-protected 2-iodoadenosine; and    -   deprotecting said N-ethylamide of said acetonide-protected        2-iodoadenosine to form said        2-iodoadenosine-5′-N-ethyluronamide.

In other embodiments, the invention is directed to methods for preparingcompounds of formula B:

-   -   wherein Z is —CH₂OC(═O)R, where R is a C₁ to C₅ alkyl,        comprising the steps of.    -   providing 1,4-methanol cyclohexane;    -   preparing a mono-tosyl derivative of said 1,4-methanol        cyclohexane;    -   preparing an acetylide-substituted compound of the formula:    -   from said mono-tosyl derivative of said 1,4-methanol        cyclohexane; and    -   converting said acetylide-substituted compound to a compound of        formula B:

In yet other embodiments, the invention is directed to methods forpreparing compounds of formula B:

-   -   wherein Z is —C(═O)OR, where R is a C₁ to C₅ alkyl, comprising        the steps of.    -   providing 1,4-methanol cyclohexane; and    -   preparing a mono-tosyl derivative of said 1,4-methanol        cyclohexane;    -   preparing an acetylide-substituted compound of the formula:    -   from said mono-tosyl derivative of said 1,4-methanol        cyclohexane;    -   oxidizing said acetylide-substituted compound using radical        oxidation; and    -   esterifying said product of said oxidation to a compound of        formula B:

In one preferred embodiment, the 2-iodoadenosine-5′-N-ethyluronamide isprepared from 2-iodoadenosine.

In another preferred embodiment, the compound of formula B is preparedfrom 1,4-methanol cyclohexane.

The term “stereoisomers,” as used herein, refers to compounds that haveidentical chemical constitution, but differ as regards the arrangementof the atoms or groups in space. It is understood that compounds offormula A may include one or more asymmetric carbons, and that formula Aencompasses all possible stereoisomers and mixtures thereof, as well asracemic modifications, particularly those that possess the activitiesdiscussed herein. Compounds prepared by the present methods may beisolated in optically active or racemic forms. Thus, all chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. Stereoisomers of the compounds offormula A can be selectively synthesized or separated in pure,optically-active form using conventional procedures known to thoseskilled in the art of organic synthesis. For example, mixtures ofstereoisomers may be separated by standard techniques including, but notlimited to, resolution of racemic forms, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

“Pharmaceutically acceptable salt,” as used herein with respect to thecompounds of the invention, refer to derivatives of the disclosedcompounds wherein the parent compound of formula A is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. These physiologically acceptable salts are prepared bymethods known in the art, e.g., by dissolving the free amine bases withan excess of the acid in aqueous alcohol, or neutralizing a freecarboxylic acid with an alkali metal base such as a hydroxide, or withan amine.

“C₁-C₅ alkyl,” as used herein, refers to an optionally substituted,saturated straight, branched, or cyclic hydrocarbon having from about 1to about 5 carbon atoms (and all combinations and subcombinations ofranges and specific numbers of carbon atoms therein), include, but arenot limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, n-pentyl, cyclopentyl, isopentyl and neopentyl.

Scheme A: Synthesis of 2-IodoNECA

One of the requirements of the methods of the invention involvesproviding the compound 2-iodoNECA. This compound may be prepared in foursteps as depicted in Scheme A.

Another method of preparing 2-iodoNECA is described in G. Cristalla etal., J. Med. Chem., 1992, 35:2363-68.

The starting material in Scheme A, 2-iodoadenosine (1), is commerciallyavailable from such sources as Toronto Research Chemicals.2-Iodoadenosine may be synthesized by the method described in Matsuda etal., J. Med. Chem., 1992, 35:241-252 and Nair et al., Synthesis, 1982,670-672.

In Step A1, 2-iodoadenosine (1) is converted to its protected form (2),preferably its acetonide-protected form, by protecting the hydroxylgroups at 2′ and 3′ carbons on the ribose ring. This protection may beaccomplished, for example, using 70% perchloric acid, methanesulfonicacid, p-toluenesulfonic acid or other sulfonic acids in excess acetonesolvent or mixtures including acetone solvent. This step may be carriedout for one hour to five hours at room temperature. The acid may beneutralized with, for example, aqueous sodium carbonate. The product (2)may be extracted in dichloromethane, for example, by concentrating thedichloromethane to solid and drying under vacuum, for example, at 40° C.This step typically provides yields as high as 90 to 93%. This improvedapproach to the rapid conversion of 2-iodoadenosine (1) to its acetonideform (2) proceeds cleanly and in high yield and requires no furtherpurification. Previously reported procedures required chromatographicseparations (WO 99/61054, Homma et al., J. Med. Chem. 1992, 35,2881-2890).

In Step A2, the primary alcohol (2) is oxidized to the acid (3). Thisstep may be carried out in about 8 to 24 hours obtaining high yieldswith simple isolation. This may be accomplished by radical oxidation,such as, for example, the use of bis-acetoxyiodobenzene with2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPO) (Epp, J. G. andWidlanski, T. S., J Org. Chem. 1999, 64, 293), hypervalent iodinespecies, derivatives of TEMPO, including 4-benzyloxy derivative of TEMPOand the like. The reaction may be carried out in solvent, such asacetonitrile, by mixing the components at ambient temperatures for about3 hours, cooling to about 0° C., filtering the resulting solids andwashing with cold solvent and then drying under vacuum under elevatedtemperatures, for example, 50° C. for about 8 to 18 hours. Typicalyields are as high as 80%. Conventional oxidation procedures usingpermanganate require long reaction times (U.S. Pat. No. 5,593,975) orhave issues with competing oxidation at the 2-iodo position (Homma etal., J. Med. Chem., 1992, 35, 2881-2890). Conventional oxidationprocedures using Ru(III) catalyst result in lower yields and requirechromatographic purification (Homma et al., J. Med. Chem., 1992, 35,2881-2890).

In Step A3, the acid (3) is converted to the N-ethylamide (4),preferably in a one pot reaction. Conventional activation methods useacid chloride. In certain embodiments, the acid may be activated as asuccinimide derivative using a carbodiimide, such asN-ethyl-dimethylaminopropylcarbodiimide (EDC). The activated acid maythen be treated with an excess of ethylamine to afford the amide in goodyield and reasonable purity. In a preferred embodiment, Step A3 may becarried out by activating the acid using2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), for example, bymixing at about 35° C. for about 3 hours and then cooling to about 5 to10° C. The reaction solution may then be directly treated with ethanoland saturated with ethylamine, to afford the N-ethylamide product (4) inhigh purity and yield after recrystallization. The recrystallization maybe carried out, for example, by first distilling the crude product (4)to a minimum volume, dissolving the residue in dichloromethane, washingwith acid and base, exchanging the dichloromethane with ethanol, coolingto 0° C. for 2 to 5 hours, filtering the product and drying at about 40°C. under vacuum for 12 to 24 hours. Typical yields are in the range of65 to 70%. Preferably, the ethylamine is added as a 4 to 8 M solution inethanol to the reaction mixture and stirred, for example, for about 16hours. Alternatively, the reaction mixture may be saturated with gaseousethylamine.

In step A4, the product (4) may be deprotected using a suitable acid,for example, trifluoroacetic acid/water or formic acid/water, to producethe key intermediate, 2-iodoNECA (5), as a pure powder that may be useddirectly in subsequent steps. This deprotection may be accomplished bymixing the reactants for about 3 to 5 hours. The compound 5 may beextracted, for example, by cooling the mixture to about 10° C. andadding to solvent, such as methyl tert-butyl ether (MTBE), stirring forabout 2 hours after the complete addition of the solvent, filtering,washing with solvent and drying under vacuum at about 40° C. Suitableacids include, for example, mineral and organic acids, with organicacids being preferred. Suitable acids include, for example, hydrochloricacid, trifluoroacetic acid and formic acid, with trifluoroacetic beingpreferred. The quantity of acid employed to deprotect the compound 4 mayvary depending, for example, on the particular acid employed, and theparticular compound 4 involved. Generally speaking, a large excess ofacid may be employed, as it is used as the solvent for the reaction. Byway of general guidance, the deprotection of compound 4 may be conductedover a wide range of temperatures. Preferably, the reaction is conductedat a temperature and for a time sufficient to form the compound offormula 5. The particular temperatures and times may vary, depending,for example, on the particular compound 4 and acid involved, as well asthe particular solvent employed. In preferred form, the deprotection ofcompound 4 may be conducted at a temperature of from about 10 to about35° C., and all combinations and subcombinations of temperature rangestherein. More preferably, the deprotection of compound 4 may beconducted at about room temperature. The yields from this step aretypically in the range of 90 to 95%.

The compound of formula 5 has been obtained in 62% overall yield fromcompound 1 with no chromatographic purification, as compared to areported 30% yield for the previous method (WO 00/44763).Scheme B1: Synthesis of cyclohexane-containing acetylene, where Z is—CH₂OC(═O)R (where R is C₁-C₅ alkyl)

Scheme B2: Synthesis of cyclohexanecontaining acetylene, where Z is—C(═O)OR (where R is C₁-C₅ alkyl)

Synthesis of the other key intermediates, the alkyne esters (10) and(12), is also improved over conventional procedures.

The starting material in Schemes B1 and B2, puretrans-cyclohexanedicarboxylic acid dimethyl ester (6), preferablycontaining less than about 0.2% by weight of the cis isomer, iscommercially available from such sources as Aldrich.

The pure trans-diol (7) may be readily obtained by reducing the diester(6) to the diol with a reducing agent, such as, for example, lithiumaluminum hydride (Step B1-1 or B2-1). The diester (6) is generally addedto the solution of reducing agent at a rate determined by keeping thetemperature of the solution between −10° C. and 25° C., preferably below10° C. Other suitable reducing agents for producing pure trans-diolinclude, for example, borane and alane agents capable of reducing estersto alcohols. In certain preferred embodiments, commercially-availablediol (7) at 98% trans isomer may be recrystallized from2-ethoxyethylacetate to levels of greater than 99.5% pure trans isomer.

In Steps B1-2 and B2-2, pure mono-tosyl derivative (8) may be preparedand isolated without additional chromatographic purification. Residualdiol starting material (7) may be removed by the aqueous workup and theditosyl product removed through selective precipitation with methanolleaving the pure mono-tosyl derivative (8) in solution. Simpleevaporation afforded the desired product (8) in pure form in 53% yield.This contrasts to the reported two-step procedure that afforded theprotected mono-tosyl derivative in 51% yield after two chromatographicpurifications (Rieger, J. M., Brown, M. L., Sullivan, G. W., Linden, J.,and MacDonald, T. L., J. Med. Chem., 2000, 44, 531-539), or in 35% yieldafter use of the single-step procedure (WO 00/44763).

It has been reported that the acetylide substitution on the tosylate isproblematic, requiring a large excess of reagent with long reactiontimes, or the use of a protected intermediate with heating (Rieger, J.M., Brown, M. L., Sullivan, G. W., Linden, J., and MacDonald, T. L., J.Med. Chem., 2000, 44, 531-539). Steps B1-3 and B2-3 of the inventionusing lithium acetylide-ethylene diamine complex and dimethylsulfoxide(DMSO) are rapid, producing high yields (90%), requiring no steps ofheating, further purification or deprotection to generate the productalcohol (9).

For cyclohexane-containing acetylene compound where Z is —C(═O)OR (whereR is C₁-C₅ alkyl) (Scheme B2), compound 9 is oxidized in step B2-4 usingstandard Jones oxidation conditions. Oxidation of the hydroxyl ofhydroxyl acetylene species using standard Jones oxidation conditions hasbeen reported to afford 70-75% yields (Rieger, J. M., Brown, M. L.,Sullivan, G. W., Linden, J., and MacDonald, T. L., J. Med. Chem., 2000,44, 531-539; WO 00/44763). Use of the TEMPO/bis-acetoxyiodobenzene(BAIB) in step B2-4 provides mild conditions for carrying out thistransformation quickly (about 3 hours) and cleanly, in high isolatedyields (95%).

In step B2-5, the carboxylic acid (11) may be converted to the desiredester (12) by a simple Fisher esterification reaction using a suitablealcohol (methanol for compound (12) or ethanol or 1-propanol), andcatalyst, such as concentrated sulfuric acid, hydrochloric acid,anhydrous hydrogen chloride, p-toluenesulfonic acid and acid form of anion exchange resin, followed by distillation, to produce the product(12) in good yield and purity without the use of expensive reagents liketrimethylsilydiazomethane. This improved synthesis affords the desiredester-alkyne in four steps in 40% overall yield with no chromatographicpurifications. This contrasts to the prior art syntheses that proceededin four steps with a 22% yield after three column chromatography steps(Rieger, J. M., Brown, M. L., Sullivan, G. W., Linden, J., andMacDonald, T. L., J. Med. Chem., 2000, 44, 531-539) or in six steps witha 28% overall yield with four chromatographic purifications (WO00/44763).

In step B1-4, trans-4-(2-propynyl)-cyclohexylmethanol 9 is converted toits acetoxymethyl derivative 10. Compound 9 is acetylated with aceticanhydride/triethylamine using 4,4-dimethylaminopyridine catalysis toafford the desired acetoxymethyl derivative 10.Scheme C1: Cross-coupling of product of Scheme B1 with product of SchemeA to produce the Compound of Formula A where Z is —CH₂OC(═O)R (where Ris C₁-C₅ alkyl)

Scheme C2: Cross-coupling of product of Scheme B2 with product of SchemeA to produce the Compound of Formula A where Z is —C(═O)OR (where R isC₁-C₅ alkyl)

Compounds of formula A of the invention may be produced by theSonagashira cross-coupling of either compound 10 with compound 5 (SchemeC1) to produce Compound A where Z is —CH₂OC(═O)R, or compound 12 withcompound 5 (Scheme C2) to produce Compound A where Z is —C(═O)OR.Generally, the halide-containing compound (either compound 10 orcompound 12) and alkyne (for example, in 50% excess) may be dissolved inan appropriate anhydrous solvent, such as dimethyl formamide orN-methylpyrrolidine, under inert headspace, preferably nitrogen orargon. The copper iodide is added, followed by the palladium catalyst,preferably bis-triphenylphosphine palladium dichloride, maintaining theinert headspace. The reaction is stirred at ambient temperatures forabout 2 to 5 hours, preferably about 2 hours, monitoring fordisappearance of the aryl halide. When the aryl halide has beenconsumed, the solvent may be replaced by dichloromethane, the solutionwashed with EDTA to remove copper, dried, filtered and concentrated. Theresidue may be purified by flash chromatography and/orrecrystallization.

The conditions for the Sonagashira coupling may be modified by the useof palladium bis-triphenylphosphine dichloride intriethylamine/dimethylformamide with no added phosphine or othercosolvent. This allows the reaction to proceed at room temperature tocompletion in 2 hours, as opposed to heating overnight as in prior artmethods. Yields after flash chromatographic purification were 76%, asopposed to a reported 24% or 60% (Rieger, J. M., Brown, M. L., Sullivan,G. W., Linden, J., and MacDonald, T. L., J. Med. Chem., 2000, 44,531-539; WO 00/44763). Even if a highly purified form is desired,preparative HPLC purification only lowers the yields to 71%.

Thus, the methods of the invention produce the desired final product ina highly pure form, preferably with <0.2% contamination by thecis-isomer impurity, in ten steps with one chromatographic purification,in an overall yield of 17.6%. All of the reported steps are amenable tosignificant scale-up to relevant manufacturing scales. This contrastswith prior art methods, which afford a 1.6% overall yield in a similarnumber of steps (Rieger, J. M., Brown, M. L., Sullivan, G. W., Linden,J., and MacDonald, T. L., J. Med. Chem., 2000, 44, 531-539), or a 5%overall yield with added steps (WO 00/44763)). In each case, asignificant number of chromatographic purifications were required, whichcreates significant issues in scaling up to a relevant manufacturingscale.

In connection with the preparation of adenosine derivatives, the methodsof the present invention may offer improved yields, purity, ease ofpreparation and/or isolation of intermediates and final product, andmore industrially useful reaction conditions and workability over priorart methods of preparation. The present methods are particularly usefulfor the preparation of adenosine derivatives on a large scale, includingcommercial scale, for example, from multi-kilogram to ton quantities ormore of adenosine derivative. Specifically, isolation and/orpurification steps of intermediates to the adenosine derivatives may beadvantageously substantially or completely avoided using the methods ofthe present invention. The present methods may be particularlyadvantageous in that the adenosine derivatives may be obtained insubstantially pure form. The term “substantially pure form”, as usedherein, means that the adenosine derivative prepared using the presentprocesses may preferably be substantially devoid of organic impurities.The term “organic impurities”, as used herein, refers to organicmaterials, compounds, etc., other than the desired product, including,for example, the cis-isomer of compound of formula A, that may betypically associated with synthetic organic chemical transformationsincluding, for example, unreacted starting reagents, unreactedintermediate compounds, and the like. In preferred form, the presentprocesses may provide adenosine compounds that are at least about 75%pure, as measured by standard analytical techniques such as, forexample, HPLC. Preferably, the adenosine derivatives prepared using thepresent methods may be at least about 80% pure, with a purity of atleast about 85% being more preferred. Even more preferably, theadenosine derivatives prepared using the present methods may be at leastabout 90% pure, with a purity of at least about 95% being morepreferred. In particularly preferred embodiments,. the adenosinederivatives prepared using the present methods may be more than about95% pure, with a purity of about 99.8% being even more preferred, andwith a purity of about 100% being especially preferred.

If a. salt of the compound of formula A is desired, a suitable acid maybe added followed by cooling and seeding of the resultant solution toprovide the crystalline salt. Preferably, the acid chosen will be ableto form the salt without affecting the integrity of the target compound.Thus, mild acids, such as sulfonic acids, are preferred. In particular,methane sulfonic acid, benzenesulfonic acid, toluenesulfonic acid,hydroxyethanesulfonic acid, camphorsulfonic acid, and other sulfonicacids may prepare suitable crystalline salts. A particularly preferredacid is methane sulfonic acid. It will be appreciated, however, thatnumerous other salts are possible, when an anhydrous form of the acid isavailable. For example, mineral acids, such as hydrochloric,hydrobromic, phosphoric, sulfuric, or nitric acid may be used to preparesuitable crystalline salts. Other organic acids, such as fumaric,succinic, oxalic, citric, and the like, may be used to prepare suitablecrystalline salts provided that they are sufficiently acidic toprotonate the basic moiety of compound of formula A.

Under appropriate conditions, however, other solvents may be used toprepare crystalline salts of formula A, such as ester solvents,including, but not limited to ethyl acetate, propyl acetate, isopropylacetate, isobutyl acetate, ethyl propionate, propyl propionate,isopropyl propionate; ether solvents, including, but not limited tot-butyl methyl ether, tetrahydrofuran, ethyl ether, isopropyl ether,butyl ether; and aromatic solvents, including, but not limited totoluene and anisole. Other solvents will be readily understood to thoseof ordinary skill in the art. Filtration and washing of the product,preferably with additional crystallization solvent, affords the compoundof formula A.

Compounds described herein throughout, can be used or prepared inalternate forms. For example, many amino-containing compounds can beused or prepared as an acid addition salt. Often such salts improveisolation and handling properties of the compound. For example,depending on the reagents, reaction conditions and the like, compoundsas described herein can be used or prepared, for example, as theirhydrochloride or tosylate salts. Isomorphic crystalline forms, allchiral and racemic forms, hydrates, solvates, and acid salt hydrates,are also contemplated to be within the scope of the present invention.

Certain acidic or basic compounds of the present invention may exist aszwitterions. All forms of the compounds, including free acid, free baseand zwitterions, are contemplated to be within the scope of the presentinvention. It is well known in the art that compounds containing bothamino and carboxyl groups often exist in equilibrium with theirzwitterionic forms. Thus, any of the compounds described hereinthroughout that contain, for example, both amino and carboxyl groups,also include reference to their corresponding zwitterions.

The reactions of the synthetic methods described and claimed herein maybe carried out in suitable solvents which may be readily selected by oneskilled in the art of organic synthesis. Generally, suitable solventsare solvents which are substantially non-reactive with the startingmaterials (reactants), the intermediates, or products at thetemperatures at which the reactions are carried out, i.e., temperatureswhich may range from the solvent's freezing temperature to the solvent'sboiling temperature. A given reaction may be carried out in one solventor a mixture of more than one solvent. Depending on the particularreaction, suitable solvents for a particular work-up following thereaction may be selected. Suitable solvents, as used herein may include,by way of example and without limitation, chlorinated solvents,hydrocarbon solvents, aromatic solvents, ether solvents, proticsolvents, polar aprotic solvents, and mixtures thereof.

Suitable halogenated solvents include, but are not limited to carbontetrachloride, bromodichloromethane, dibromochloromethane, bromoform,chloroform, bromochloromethane, dibromomethane, butyl chloride,dichloromethane, tetrachloroethylene, trichloroethylene,1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane,2-chloropropane, hexafluorobenzene, 1,2,4-trichlorobenzene,o-dichlorobenzene, chlorobenzene, fluorobenzene, fluorotrichloromethane,chlorotrifluoromethane, bromotrifluoromethane, carbon tetrafluoride,dichlorofluoromethane, chlorodifluoromethane, trifluoromethane,1,2-dichlorotetrafluorethane and hexafluoroethane.

Suitable hydrocarbon solvents include, but are not limited to alkane oraromatic solvents such as cyclohexane, pentane, hexane, toluene,cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o-, orp-xylene, octane, indane, nonane, benzene, ethylbenzene, and m-, o-, orp-xylene.

Suitable ether solvents include, but are not limited todimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan,diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethylether, diethylene glycol dimethyl ether, diethylene glycol diethylether, triethylene glycol diisopropyl ether, anisole, or t-butyl methylether.

Suitable protic solvents include, but are not limited to water,methanol, ethanol, 2-nitroethanol, 2-fluoroethanol,2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol,2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butylalcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3- pentanol,neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethylether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol,phenol, and glycerol.

Suitable aprotic solvents include, but are not limited todimethylformamide (DMF), dimethylacetamide (DMAC),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP),formamide, N-methylacetamide, N-methylformamide, acetonitrile (ACN),dimethylsulfoxide (DMSO), propionitrile, ethyl formate, methyl acetate,hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate,isopropyl acetate, t-butyl acetate, sulfolane (tetramethylene sulfone),N,N-dimethylpropionamide, nitromethane, nitrobenzene, andhexamethylphosphoramide.

The invention is further described in the following examples. All of theexamples are actual examples. These examples are for illustrativepurposes only, and are not to be construed as limiting the appendedclaims.

EXAMPLES Example 1

Synthesis of[(1R,2R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7,7-dimethyl-3,6,8-trioxabicyclo[3.3.0]oct-2-yl]methan-1-ol (Compound 2)

To a suspension of 2-iodoadenosine 1 (10.0 g, 25.4 mmol) in acetone (200ml) cooled to 0° C. was added dropwise 70% perchloric acid (4.0 mL),resulting in an exotherm of about 5° C. The resultant colorless solutionwas allowed to warm to room temperature over 30 minutes, then stirredfor a further 45 minutes. 1M Na₂CO₃ (50 mL) was added, resulting insolids precipitating. This was followed by the careful portionwiseaddition of water (300 mL) with stirring until all solids had dissolved.The mixture was extracted with three portions of CH₂Cl₂. The combinedorganics were washed with brine, dried (Na₂SO₄), filtered, andevaporated to afford Compound 2 (10.26 g, 93%) as a colorless solid.¹H-NMR (600 MHz, DMSO d₆): 1.32 (s, 3H), 1.54 (s, 3H), 3.54 (m, 2H),4.19 (m, 1H), 4.93 (dd, 1H), 5.05 (t, 1H), 5.27 (dd, 1H), 6.05 (d, 1H),7.74 (bs, 2H), 8.28 (s, 1H); ¹³C-NMR (150 MHz, DMSO d₆): 25.45, 27.08,85.69, 86.09, 88.44, 92.59, 114.92, 120.39, 142.47, 151.08, 157.20,173.14. LRMS (ES): m/z=434.0 (M+H, 100%).

Example 2

Synthesis of1′-deoxy-1′-(6-amino-2-iodo-9H-purin-9-yl)-2′,3′-O-isopropylidene-β-D-ribofuranuronicacid (Compound 3)

To a solution of Compound 2 (10.0 g, 23.1 mmol) in CH₃CN (200 mL) andwater (50 mL) cooled to 0° C. was added iodobenzene diacetate (16.4 g,50.8 mmol) and TEMPO (0.72 g, 20 mmol). The mixture was stirred at 0° C.for 30 minutes, then allowed to warm to room temperature and stirred for22 h. The solvents were evaporated and the resulting residue wastriturated with n-heptane (400 mL) overnight. The solids were filtered,washed with n-heptane and dried in vacuo to afford Compound 3 (9.80 g,95%) as an off-white solid. ¹H-NMR (600 MHz, CD₃OD): 1.45 (s, 3H), 1.65(s, 3H), 4.78 (d, 1H), 5.50 (d, 1H), 5.67 (dd, 1H), 6.31 (s, 1H), 8.14(s, 1H); ¹³C-NMR (150 MHz, CD₃OD): 25.45, 27.08, 85.69, 86.09, 88.44,92.59, 114.92, 120.39, 142.47, 151.08, 157.20, 173.14. LRMS (ES): 448.0(M+H, 100%).

Example 3

Synthesis ofN-ethyl-1deoxy-1′-(6-amino-2-iodo-9H-purin-9-yl)-2′,3′-O-isopropylidene-β-D-ribofuranuronamide(Compound 4)

To a solution of Compound 3 (8.0 g, 17.9 mmol) in 50% ethanol/CH₂Cl₂(160 mL) was added 2-ethoxy-1-ethocycarbonyl-1,2-dihydroquinoline (EEDQ)(4.65 g, 18.8 mmol) as a single portion. The mixture was stirred at roomtemperature for 24 hours. Ethanol (80 mL) was added and ethylamine gas(≈45 g) bubbled through the reaction solution over 4 hours. The reactionwas stirred at room temperature for 20 hours after which the solventswere evaporated. The resulting solids were dissolved in CH₂Cl₂ andwashed successively with 0.1 M HCl and 1M Na₂CO₃. The organics weredried (Na₂S0₄), filtered, and evaporated to afford Compound 4 as a paleyellow solid which was recrystallized from CH₂Cl₂/hexanes to affordCompound 4 (6.0 g, 71%) as a white solid, m.p. 204-206° C.; ¹H-NMR (600MHz, DMSO-d₆): 0.68 (t, 3H), 1.32 (s, 3H), 1.52 (s, 3H), 2.81 (m, 1H),2.91 (m, 1H), 4.53 (s, 1H), 5.33 (m, 1H), 5.36 (m, 1H), 6.27 (s, 1H),7.43 (t, 1H), 7.68 (s, 2H), 8.16 (s, 1H); ¹³C-NMR (150 MHz, DMSO-d₆):14.07, 25.06, 26.61, 33.07, 83.15, 83.35, 112.77, 118.71, 120.68,139.99, 149.31, 155.81, 167.92. LRMS (ES): m/z=475.0 (M+H, 100%).

Example 4

Synthesis of[(2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihyroxyoxolan-2-yl]-N-ethylcarboxamide(Compound 5)

To a stirred solution of 10% aqueous trifluoroacetic acid (1100 mL) isadded Compound 4 (80 g, 168 mmol) over 5 minutes with stirring. Thesolution was stirred for 1 hour at room temperature, and thenconcentrated under reduced pressure. The resulting brown oil wastriturated with methyl tert-butyl ether (MTBE) to afford a solid whichwas filtered, rinsed with MTBE, and dried under vacuum to yield Compound5 (72.7 g, 99%) as a white powder. ¹H-NMR (600 MHz, DMSO-d₆): 1.05 (t,3H), 3.24 (m, 2H), 4.17 (dd, 1H), 4.31 (d, 1H), 4.58 (dd, 1H), 5.91 (d,1H), 7.74 (bs, 2H), 8.10 (t, 1H), 8.38 (s, 1H); ¹³C-NMR (150 MHz,DMSO-d6): 14.78, 26.77, 33.47, 72.65, 72.94, 84.20, 119.16, 120.91,149.84, 155.90, 169.00; LRMS (ES): m/z=435.1 (M+H, 100%) Example 5

Synthesis of trans-[(4-hydroxymethyl) cyclohexyl]methan-1-ol (Compound7)

To a suspension of lithium aluminum hydride (7.6 g, 0.2 mol) inanhydrous tetrahydrofuran (600 mL) cooled to 4° C. under a positivenitrogen pressure was added dropwise over 30 minutes a solution oftrans-1,4-dimethyl-cyclohexanedicarboxylate 6 (20 g, 0.1 mol) inanhydrous tetrahydrofuran (400 mL) at a rate to maintain a temperatureof <10° C. during addition. The reaction was stirred with cooling for afurther 30 minutes, then allowed to warm to room temperature and stirredfor 70 hours. The reaction was cooled to 4° C., and water (7.6 mL) addedcarefully, followed by 15% NaOH (7.6 mL), and water (22.8 mL). Themixture was allowed to gradually warm to room temperature, then stirredat room temperature for 5 hours. The resultant colorless suspension wasfiltered, and the filtrate concentrated to afford Compound 7 (13.0 g,90%) as an off-white solid. ¹H-NMR (400 MHz, CDCl₃): 0.83 (m, 4H), 1.26(m, 2H), 1.72 (d, 4H), 3.19 (t, 4H), 4.34 (t, 2H); ¹³C-NMR (100 MHz,CDCl₃): 29.05, 40.63, 66.79.

Example 6

Synthesis of trans-[{(4-hydroxymethyl)cyclohexyl}methyl]4-methylbenzenesulfonate (Compound 8)

To a solution of Compound 7 (100 g, 0.69 mol) in anhydrous pyridine (1L) stirred at room temperature was added portionwisep-toluenesulfonylchloride (132 g, 0.69 mol). The reaction was stirredfor 1 hour, over which time a colorless precipitate was observed toform. The reaction temperature was cooled to −10° C. and water (4 Ltotal) added carefully, keeping the temperature <20° C. The mixture wasextracted with CH₂Cl₂, washed with 3M HCl, dried (MgSO₄), filtered, andevaporated. The crude residue was triturated with anhydrous MeOH and theresulting solids were removed by filtration. The filtrate was evaporatedto afford Compound 8 (110 g, 53%) as a pale yellow oil. ¹H-NMR (400 MHz,CDCl₃): 0.91 (m, 4H), 1.37 (m, 1H), 1.58 (m, 1H), 1.75 (m, 4H), 1.84,(s, 1H), 2.42 (s, 3H), 3.39 (d, 2H), 3.80 (d, 2H), 7.34 (d, 2H), 7.75(d, 2H); ¹³C-NMR (100 MHz, CDCl₃): 21.53, 28.32, 28.34, 37.22, 39.99,68.07, 75.16, 127.73, 129.72, 132.85, 144.62.

Example 7

Synthesis of (4-prop-2-ynylcyclohexyl)methan-1-ol (Compound 9)

To a solution of Compound 8(110 g, 0.37 mol) in DMSO (1.46 L) was addedas a single portion, lithium acetylide/ethylenediamine complex (>90%purity, 101.4 g, 1.10 mol). The reaction was stirred at ambienttemperature for 2 hours. Water (3.8 L) was then added cautiously over 35minutes, maintaining a solution temperature <37° C. Methyl tert-butylether (MTBE) (3 L) was then added and the biphasic mixture stirredvigorously for 10 minutes. The organic phase was separated and theaqueous phase further extracted with MTBE. The combined extracts werewashed with water, dried (MgSO₄), filtered, and evaporated to affordCompound 9 (50.3 g, 90%) as a pale yellow oil. ¹H-NMR (400 MHz, CDCl₃):0.96 (m, 4H), 1.41 (m, 2H), 1.82 (m, 4 H), 1.94, (t, 1H), 2.07 (d of d,2H), 2.16 (s, 1H), 3.39 (d, 2H); ¹³C-NMR (100 MHz, CDCl₃): 25.91, 29.04,31.71, 37.04, 40.07, 68.23, 69.01, 83.24.

Example 8

Synthesis of trans-4-(2-propynyl)-cyclohexylmethanol acetate (Compound10)

A solution of trans-4-(2-propynyl)-cyclohexylmethanol (Compound 9) (15g, 98 mmol), triethylamine (TEA) (15.1 mL, 108 mmol), and4-(N,N-dimethyl-amino)pyridine (DMAP) (0.6 g, 5 mmol) in dichloromethane(300 mL) was cooled to 2° C. with stirring under nitrogen. Aceticanhydride (Ac₂O) (10.2 mL, 108 mmol) was added dropwise, keepingtemperature below 10° C. After 30 minutes stirring at ice bathtemperature, the reaction was quenched with 10% potassium carbonatesolution (300 mL). The layers were separated and the organic layer waswashed successively with 1N HCl, water, and brine, dried over magnesiumsulfate, filtered, and concentrated under vacuum to afford 20 g ofproduct as a yellow oil which was distilled bulb to bulb under vacuum(0.5 torr, 70° C.) to afford the product (Compound 10) as a colorlessoil (17 g, 89%). ¹H-NMR (600 MHz, CDCl₃): 1.03 (m, 4H), 1.46 (m, 1H),1.59 (m, 1H), 1.85 (dd, 4H), 1.89 (t, 1H), 2.05 (t, 3H), 2.11 (dd, 2H),3.89 (d, 2H).

Example 9

Synthesis of 4-prop-2-ynylcyclohexane carboxylic acid (Compound 11)

To a suspension of Compound 9 (2.45 g, 16 mmol) in 50%acetonitrile/water (35 mL) cooled to 0° C. was addedbis-acetoxy-iodobenzene (BAIB) (11.4 g, 35 mmol) and TEMPO (502 mg, 3.2mmol). The reaction mixture was stirred at 0° C. for 1 hour, then for 90minutes at room temperature. The solvents were evaporated, and theresidue dissolved in 1/1 CH₃CN/H₂O (100 mL). Once again the solventswere evaporated, and this procedure repeated to remove iodobenzene asits azeotrope. The resulting Compound 11 (2.53 g, 95%) was obtained as abrown sticky solid that was not further purified but carried forwardinto the next step. ¹H-NMR (600 MHz, CD₃OD): 1.13 (dq, 2H), 1.45 (dq,2H), 1.48 (m, 1H), 1.93 (dd, 2H), 2.03 (dd, 2H), 2.12 (dd, 2H), 2.2-2.3(m, 2H).

Example 10

Synthesis of methyl 4-prop-2-ynyl cyclohexanecarboxylate (Compound 12)

To a solution of Compound 11 (0.88 g, 5.29 mmol) in methanol (10 mL) wasadded concentrated HCl (0.5 mL) and the mixture stirred at roomtemperature for 2 hours. The solvents were evaporated and the residuedissolved in CH2Cl₂, dried (Na₂SO₄), filtered, and evaporated to afforda pale yellow oil. The oil was purified by bulb-to-bulb distillation(140° C. at 500 mTorr) to afford Compound 12 (0.80 g, 84%) as a palepink oil. ¹H-NMR (600 MHz, CD₃OD): 1.13 (dq, 2H), 1.45 (dq, 2H), 1.48(m, 1H), 1.94 (dd, 2H), 2.01 (dd, 2H), 2.12 (dd, 2H), 2.24 (t, 1H), 2.28(m, 1H), 3.68 (s, 3H); ¹³C-NMR (150 MHz, CD₃OD): 26.55, 29.96, 32.48,37.79, 44.31, 52.08, 70.57, 83.44; FTIR (KBr) 1743 (C═O), 2117 (C≡C),2872, 2953 (C—H), 3314 cm⁻¹ (C≡C—H)

Example 11

1-[2-[3-[trans-4-[(Acetyloxy)methyl]cyclohexyl]-1-propynyl]-6-amino-9H-purin-9-yl]-1-deoxy-N-ethyl-β-D-ribofuranuronamide(Compound A where Z is —CH₂OC(═O)CH₃)

To a stirred suspension of1-(6-amino-2-iodo-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide5 (75 mg, 0.17 mmol) in dry dioxane (2 mL) was addedtrans-4-(2-propynyl)-cyclohexylmethanol acetate 10 (54 mg, 0.28 mmol),triethylamine (78 μL, 0.56 mmol), copper iodide (11.3 mg, 59 μmol),bis-acetonitrile palladium dichloride (11.8 mg, 45 μmol), andtri(tert-butyl)phosphine (10% in hexanes, 93 μL, 30 μmol). This wasstirred for 20 hours at room temperature, then triethylamine (1 mL) wasadded and the solution heated to 50° C. for 5 hours. Dimethylformamide(2 mL) was added along with additional aliquots of cyclohexylmethanolacetate (100 mg), CuI (5 mg), Pd(MeCN)₂Cl₂ (10 mg), andtri(tert-butyl)phosphine (200 μL), and the heating continued for 26hours. The solvents were removed under vacuum, and the residue dissolvedin chloroform, washed with 0.01 M Na₂EDTA solution (2×50 mL) and brine,dried over sodium sulfate, filtered, and evaporated. The residue waspurified by flash chromatography (95:5 dichloromethane/methanol) toafford the product (Compound A where Z is —CH₂OC(═O)CH₃) as a brownsolid (40 mg, 47%): ¹H-NMR (600 MHz, DMSO-d₆): 1.01 (m, 4H), 1.06 (t,3H), 1.48 (m, 2H), 1.8 (dd, 4H), 2.00 (s, 3H), 2.32 (d, 2H), 3.25 (m,1H), 3.30 (m, 1H), 3.83(d, 2H), 4.11 (t, 1H), 4.30 (d, 1H), 4.57 (m,1H), 5.53 (d, 1H), 5.73 (d, 1H), 5.92 (d, 1H), 7.52 (bs, 2H) 8.41 (s,1H), 8.73 (t, 1H); ¹³C-NMR (150 MHz, CD₃OD): 14.94, 25.67, 28.21, 31.30,33.28, 36.38, 36.50, 68.52, 71.80, 73.11, 81.93, 84.38, 84.65, 87.64,119.06, 141.45, 145.63, 148.95, 155.96, 169.17, 170.38; LRMS (ES): 501.2(M+H, 100%).

Example 12

1-[2-[3-[trans-4-[(Acetyloxy)methyl]cyclohexyl]-1-propynyl]-6-amino-9H-purin-9-yl]-1-deoxy-N-ethyl-β-D-ribofuranuronamide(Compound A where Z is —CH₂OC(═O)CH₃)

Alternatively, Compound A where Z is —CH₂OC(═O)CH₃ may be obtained bythe following procedure:

A flask is charged with Compound 5 (33 g, 76 mmol), Compound 10 (22.3 g,115 mmol), dry dimethylformamide (330 mL), and triethylamine (115 mL)under a nitrogen atmosphere. The stirred solution is sparged with drynitrogen for 15 minutes and then copper iodide (2.21 g, 11.6 mmol) andpalladium bis(triphenylphosphine)dichloride (4 grams, 5.7 mmol) areadded, maintaining nitrogen purge. The reaction is stirred at roomtemperature for 1.5 hours and concentrated under reduced pressure toproduce a brown oil. This is redissolved in dichloromethane and washedwith four portions of 0.1N EDTA solution. The organics are dried(Na₂SO₄), filtered, and concentrated under vacuum. The crude material ispurified by flash chromatography (1-7% methanol gradient indichloromethane) to afford Compound A where Z is —CH₂OC(═O)CH₃ as a paleyellow solid.

Example 13

Synthesis of 4-{3-[6-amino-9-(5-ethyl carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclo hexanecarboxylicacid methyl ester (Compound A where Z is —C(═O)OCH₃)

A flask was charged with Compound 5 (33 g, 76 mmol), Compound 12 (20.7g, 115 mmol), dry dimethylformamide (330 mL), and triethylamine (115 mL)under a nitrogen atmosphere. The stirred solution was sparged with drynitrogen for 15 minutes and then copper iodide (2.21 g, 11.6 mmol) andpalladium bis(triphenylphosphine)dichloride (4 grams, 5.7 mmol) wereadded, maintaining nitrogen purge. The reaction was stirred at roomtemperature for 1.5 hours and concentrated under reduced pressure to abrown oil. This was redissolved in dichloromethane and washed with fourportions of 0.1N EDTA solution. The organics were dried (Na₂SO₄),filtered, and concentrated under vacuum. The crude material was purifiedby flash chromatography (1-7% methanol gradient in dichloromethane) toafford Compound A where Z is —C(═O)OCH₃ (28.1 grams, 76%) as a paleyellow solid.

Example 14

Purification of 4-{3-[6-amino-9-(5-ethyl carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclohexane carboxylicacid methyl ester (Compound A where Z is —C(═O)OCH₃)

Purification of Compound A where Z is —C(═O)OCH₃ was accomplished in twoways: preparative HPLC and recrystallization. Preparative HPLC wascarried out on a Dynamax C-18 (8 μm, 60 A, 15×30 cm) using gradient of25%-40% B over 60 minutes (A=0.05N ammonium acetate, pH=5.0;B=acetonitrile) with an injection solution of 22.8 g Compound A where Zis —C(═O)OCH₃ in 6 L of 25% acetonitrile in 0.05 N ammonium acetate. Theeluent was monitored at 270 nM and product fractions isolated. Theproduct fractions were combined and diluted with an equal volume ofwater. They were injected onto the same column running a gradient of20%-80% B over 30 minutes (A=water; B=acetonitrile) to remove salts.Product fractions were combined, frozen, and lyophilized to affordCompound A where Z is —C(═O)OCH₃ (21.4 g, 94%) as a flocculent whitepowder. Crystallization were done from 10:1 2-propanol/methyl tert-butylether (MTBE). Compound A where Z is —C(═O)OCH₃ was dissolved in aminimum volume of hot 2-PrOH and MTBE added while hot. The solution wasallowed to cool with stirring in a 0° C. bath. It was allowed to standovernight at −8° C. and then filtered, washed with 1:1 2-PrOH/MTBE,followed by MTBE, and then dried under vacuum to afford Compound A whereZ is —C(═O)OCH₃ as a white flowable powder. ¹H-NMR (600 MHz, CD₃OD):1.21 (t, 3H), 1.22 (dq, 2H), 1.48 (dq, 2H), 1.65 (m, 1H), 2.04 (dt, 4H),2.34 (dt, 1H), 2.45 (d, 2H), 3.44 (dm, 2H), 3.69 (S, 3h), 4.36 (dd, 1H),4.50 (d, 1H), 4.74 (dd, 1H), 6.07 (d, 1H, 8.48 (s, 1H); ¹³C-NMR (150MHz, CD₃OD): 14.78, 26.77, 33.47, 72.65, 72.94, 84.20, 119.16, 120.91,149.84, 155.90, 169.00; LRMS (ES): 487.2 (M+H, 100%).

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1. A method for preparing a compound of formula A:

or a stereoisomer, pharmaceutically acceptable salt, hydrate, solvate,acid salt hydrate or isomorphic crystalline form thereof, the methodcomprising the step of: contacting 2-iodoadenosine-5′-N-ethyluronamidewith a compound of formula B:

wherein Z is —C(═O)OR or —CH₂OC(═O)R, where R is a C₁ to C₅ alkyl.
 2. Amethod according to claim 1, wherein Z is —C(═O)OCH₃ or —CH₂OC(═O)CH₃.3. A method according to claim 1, wherein said2-iodoadenosine-5′-N-ethyluronamide is prepared from 2-iodoadenosine. 4.A method according to claim 3, further comprising the step of convertingsaid 2-iodoadenosine to the acetonide-protected form of said2-iodoadenosine of the formula:


5. A method according to claim 4, further comprising the step ofoxidizing said acetonide-protected form of said 2-iodoadenosine to acompound of the formula:


6. A method according to claim 5, wherein said oxidizing step is aradical oxidation.
 7. A method according to claim 5, wherein saidoxidizing step comprises the use of a mixture comprisingbis-acetoxyiodobenzene and 2,2,6,6-tetramethyl piperidinyloxy freeradical (TEMPO).
 8. A method according to claim 5, further comprisingthe step of converting said compound of the formula:

to a compound of the formula:


9. A method according to claim 8, wherein said converting step proceedsas a one pot reaction.
 10. A method according to claim 8, wherein saidconverting step comprises the use of a succinimide derivative and anexcess of ethylamine.
 11. A method according to claim 8, wherein saidconverting step comprises the use of a composition comprising2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ethanol and ethylamine.12. A method according to claim 8, further comprising the step ofdeprotecting said compound of the formula:

to form said 2-iodoadenosine-5′-N-ethyluronamide.
 13. A method accordingto claim 1, wherein said compound of formula B is prepared from1,4-methanol cyclohexane.
 14. A method according to claim 13, whereinsaid 1,4-methanol cyclohexane is prepared from a reaction mixturecomprising trans-1,4-dimethyl-cyclohexanedicarboxylate.
 15. A methodaccording to claim 14, wherein said reaction mixture comprises less thanabout 0.2% by weight, based on the total weight of the1,4-dimethyl-cyclohexanedicarboxylate, ofcis-1,4-dimethyl-cyclohexanedicarboxylate.
 16. A method according toclaim 13, wherein said 1,4-methanol cyclohexane is prepared by a processcomprising the step of reducing 1,4-dimethyl-cyclohexanedicarboxylate.17. A method according to claim 13, further comprising the step ofpreparing the mono-tosyl derivative of said 1,4-methanol cyclohexane.18. A method according to claim 17, further comprising the step ofacetylide substitution of said tosyl derivative of the formula:

to form an acetylide-substituted compound of the formula:


19. A method according to claim 18, further comprising the step ofconverting said acetylide-substituted compound to a compound of formulaB, where Z is —CH₂OC(═O)R.
 20. A method according to claim 18, furthercomprising the steps of oxidizing said acetylide-substituted compoundand esterifying said product of said oxidation to a compound of formulaB, where Z is —C(═O)OR.
 21. A method for preparing2-iodoadenosine-5′-N-ethyluronamide, comprising the steps of: providing2-iodoadenosine; protecting the hydroxyl groups of said 2-iodoadenosinewith an acetonide group; oxidizing the primary alcohol of saidacetonide-protected 2-iodoadenosine to an acid derivative of saidacetonide-protected 2-iodoadenosine; converting said acid derivative toan N-ethylamide derivative of said acetonide-protected 2-iodoadenosine;and deprotecting said N-ethylamide of said acetonide-protected2-iodoadenosine to form said 2-iodoadenosine-5′-N-ethyluronamide.
 22. Amethod for preparing a compound of formula B:

wherein Z is —CH₂OC(═O)R, where R is a C₁ to C₅ alkyl, comprising thesteps of. providing 1,4-methanol cyclohexane; and preparing a mono-tosylderivative of said 1,4-methanol cyclohexane; preparing anacetylide-substituted compound of the formula:

from said mono-tosyl derivative of said 1,4-methanol cyclohexane; andconverting said acetylide-substituted compound to a compound of formulaB:


23. A method for preparing a compound of formula B:

wherein Z is —C(═O)OR, where R is a C₁ to C₅ alkyl, comprising the stepsof. providing 1,4-methanol cyclohexane; preparing a mono-tosylderivative of said 1,4-methanol cyclohexane; preparing anacetylide-substituted compound of the formula:

from said mono-tosyl derivative of said 1,4-methanol cyclohexane;oxidizing said acetylide-substituted compound using radical oxidation;and esterifying said product of said oxidation to a compound of formulaB:


24. A method according to claim 23, wherein said oxidizing stepcomprises the use of a mixture comprising bis-acetoxyiodobenzene and2,2,6,6-tetramethyl piperidinyloxy free radical (TEMPO).
 25. A methodaccording to claim 22 or claim 23, wherein said 1,4-methanol cyclohexaneis prepared from a reaction mixture comprisingtrans-1,4-dimethyl-cyclohexanedicarboxylate.
 26. A method according toclaim 25, wherein said reaction mixture comprises less than about 0.2%by weight, based on the total weight of the1,4-dimethyl-cyclohexanedicarboxylate, ofcis-1,4-dimethyl-cyclohexanedicarboxylate.
 27. A method according toclaim 22 or claim 23, wherein said 1,4-methanol cyclohexane is preparedby a process comprising the step of reducing1,4-dimethyl-cyclohexanedicarboxylate.